Sustainable process for the treatment and detoxification of liquid waste

ABSTRACT

A method for treatment of liquid waste is disclosed that includes the steps of (a) submitting the liquid waste to a pretreatment and (b) submitting the pretreated liquid waste to the action of fungi or active agents thereof. In particular, the described process is useful for the effective decoloration and detoxification of dye-containing liquid wastes using white rot fungi or active agents thereof.

FIELD OF THE INVENTION

The present invention relates to a sustainable process for the treatmentof and detoxification of liquid waste. In particular the inventionrelates to a process for the effective decoloration and detoxificationof liquid waste containing dyes.

BACKGROUND OF THE INVENTION

A great variety of synthetic dyes are used in numerous industries fortextile and leather dyeing, paper printing, color photography, and asadditives in petroleum and cosmetic products. Their structural diversityderives from the use of different chromophoric groups such as azogroups, anthraquinonic groups, etc. The total world colorant productionis estimated to be in the region of 800,000 tons/year. During the dyeingprocess up to 15% of the dyes are released into liquid industrialwastes. Most of these compounds are highly resistant to microbial attackand are hardly removed from effluents by conventional biological,physical or chemical treatments. In addition to visual pollution, thereis a considerable risk of toxicity toward living organisms, possibly dueto derivatives generated during bio-transformation. Textile dyes resistfading upon exposure to sweat, light, water and oxidizing agents. Theyare very stable and difficult to degrade. They are not degraded neitherby activated sludge nor by aerobic bacterial isolates. Reductiveanaerobic cleavage of these dyes results in carcinogenic compounds beinggenerated. In other words, there is a problem of water pollution fromthe dye complexes, which are discharged into public water supplies.These organic substances render the effluents highly colored and makethem toxic.

Bio-decoloration of lignin-containing pulp and paper wastewater usingwhite-rot fungi Phanerochaete chrysosporium and Tinctoporia sp. (Eaton,et al., 1980; Fukuzumi, et al., 1980) were clear examples of colorremoval through microbial degradation of the colored substances, i.e.,highly chlorinated and oxidized polymeric lignin molecules. As for dyecolor removal, Groff and Kim, 1989, described the ability ofRhodococcus, Bacillus cereus and Plesiomonas/Achromobacter to degradesoluble dyes, acid red dye and five azo-dyes, respectively.

Above reviewed bio-decoloration reports limit their studies primarily indefined laboratory model systems. Their actual application andeffectiveness towards highly colored industrial liquid waste were notparticularly emphasized. Though limited, successful examples ofbio-decoloration of pulp and paper wastewater using white-rot fungi(U.S. Pat. No. 4,655,926) were reported. U.S. Pat. No. 5,091,089 furtherdiscloses a biological approach through the use of white-rot fungi forthe decoloration of dye wastewater. The former demonstrated the use of arotating biological contactor and strains of white-rot fungi from thegenus Myrothecium and the genus Ganoderma to remove color in wasteliquor without giving quantitative results, while the latter claimed asubstantial color removal through a mechanism of dye adsorption.However, in some environmental legislation, adsorption is considered asa pollution transfer since the xenobiotic molecules are not destroyedbut are concentrated and must be transferred into dumping grounds.

There is a need to substantially remove the color from industrialeffluents and to further detoxify certain refractory organic compoundscontained in said effluents.

It is a main object of the invention to provide with a process for thetreatment of liquid waste. It is another object to provide with aprocess, which permits the effective decoloration of liquid waste. It isa further object of the invention to provide with a process, whichpermits the effective detoxification of said liquid waste. It is yet afurther object of the invention to provide with a process, which permitsthe reduction of the mutagenicity of said liquid waste.

SUMMARY OF THE INVENTION

The present invention is related to a unique and effective process toefficiently decolorize and detoxify liquid waste such as industrialeffluents. The present invention relates to a process for the treatmentof liquid waste, comprising the steps of (a) submitting said liquidwaste to a pretreatment and (b) submitting said pretreated liquid wasteto the action of fungi or active agents thereof. This process permitsthe effective decoloration, but also simultaneously the efficientdetoxification of said liquid waste. Moreover, the combination of saidpretreatment with a treatment with white-rot fungi or active agentsthereof, increases the biodegradability of said liquid waste, andtotally eliminates the mutagenicity of said liquid waste.

In an embodiment, the invention is related to a process for thetreatment of liquid waste, comprising the steps of

-   a) submitting said liquid waste to a pretreatment,-   b) submitting said pretreated liquid waste to the action of    white-rot fungi or active agents thereof.

In another preferred embodiment, the white-rot fungi active agentscomprise hydrolytic enzymes, cellulolytic enzymes, or ligninolyticenzymes.

In a more preferred embodiment said white-rot fungi active agentsconsist essentially of laccase enzymes.

In a more preferred embodiment said liquid waste, is dye containingliquid waste, comprising azo dyes and anthraquinones dyes In anothermore preferred embodiment said liquid waste comprises humic acids.

A possible pretreatment is ozonisation. Another preferred pretreatmentis the adsorption of said waste on a biodegradable support.

In an embodiment, said fungi are lignicolous fungi, more preferablywhite-rot fungi. In yet another embodiment said fungi are selected fromthe group consisting of the genus Acanthophysium, the genus Aleurobotrysthe genus Aleurodiscus, the genus Amphinema, the genus Amylostereum, thegenus Armillaria, the genus Aspergillus, the genus Asterostroma, thegenus Auricularia, the genus Botryobasidium, the genus Botryohypochnus,the genus Calocera, the genus Chaetomium, the genus Cladorrhinum, thegenus Clitocybula, the genus Columnocystis, the genus Coriolopsis, thegenus Cystostereum, the genus Daedalea, the genus Daedaleopsis, thegenus Dichomitus, the genus Dichostereum, the genus Echinodontium, thegenus Fibulomyces, the genus Fomitopsis, the genus Fusarium, the genusGanoderma, the genus Grifola, the genus Hapalopilus, the genus Humicola,the genus Hymenochaete, the genus Hyphoderma, the genus Hyphodontia, thegenus Hypochnicium, the genus Inonotus, the genus Irpex, the genusLaurilia, the genus Laxitextum, the genus Lentinus, the genus Lenzites,the genus Lentinula, the genus Leucogyrophana, the genus Lycoperdon, thegenus Marasmius, the genus Merulius, the genus Mycoacia, the genusMyrothecium, the genus Paecilomyces the genus Panellus, the genusPenicillium, the genus Peniophora, the genus Perenniporia, the genusPestalotia, the genus Phanerochaete, the genus Phellinus, the genusPhlebia, the genus Pholiota, the genus Pleurotus, the genus Polyporus,the genus Poda, the genus Punctularia, the genus Pycnoporus, the genusResinicium, the genus Schizophyllum, the genus Scytinostroma, the genusteccherinum, the genus Trametes, the genus Trichoderma, the genusTyromyces and the genus Vararia.

In a more preferred embodiment said white-rot fungi are selected fromthe group consisting of the genus Acanthophysium, the genus Coriolopsis,the genus Clitocybula, the genus Cystostereum, the genus Ganoderma, thegenus Paecilomyces, the genus Perenniporia, the genus Phellinus, thegenus Phlebia, the genus Pycnoporus, and the genus Trametes.

In yet a more preferred embodiment said white-rot fungi are selectedform the group consisting of Acantophysium bisporum MUCL 32213,Coriolopsis polyzona MUCL 38443, Cystostereum murraii MUCL 33747,Ganoderma subamboinense MUCL 38859, Lentinus cladopus MUCL 28678,Lentinula edodes MUCL 29756, Lenzites betulina MUCL 38559, Meruliustremelosus MUCL 38065, Paecilomyces variotii MUCL 21705, Perenniporiamedulla-panis MUCL 40050, Perenniporia ochroleuca MUCL 41114,Perenniporia tephropora MUCL 41562, Phanerochaete chrysospodum MUCL19343, Phanerochaete ericina MUCL 33845, Phellinus rimosus MUCL 38446,Phlebia subserialis MUCL 33724, Polyporus brumalis MUCL 29280, Polyporusciliatus MUCL 40141, Pycnoporus cinnabarinus MUCL 38520, Pycnoporuscoccineus MUCL 38525, Pycnoporus sanguineus MUCL 41625, Trametesversicolor MUCL 38412 and MUCL 28407.

In yet another more preferred embodiment said white-rot fungi areselected from the group consisting of Clitocybula dusenii DSM 11238,Trichoderma harzanium MUCL 29707 and Trichoderma longibrachiatum MUCL39887.

In a preferred embodiment said white-rot fungi is grown in a mediacontaining malt extract in a concentration ranging from 0.5 to 8 percentby weight to volume.

In another preferred embodiment said white-rot fungi are added to saidliquid waste in an encapsulated form, in a matrix consisting ofpolymers.

In a preferred embodiment said polymers are selected from the groupconsisting of alginate salts, carrageenan salts, iota-carrageenan salts,maltodextrin, whey protein concentrate (WPC), skimmed milk powder (SMP),dried yeast autolysate (YA), dried yeast extract (YE), corn starch (CS),modified starch (MS), and polyvinylalcohol.

In a preferred embodiment said white-rot fungi, are employed in animmobilized form.

In a more preferred embodiment said white-rot fungi are immobilized on asupport selected from the group consisting of stainless steel support,polymer support and wood support.

In another preferred embodiment, the white-rot fungi active agents areemployed as raw preparation, as purified enzymes, or in an immobilizedform.

In yet another more preferred embodiment, the white-rot fungi activeagents are immobilized on a wood support.

In a preferred embodiment an inductor is added to the white-rot fungiculture, preferably after said fungi has reached a significant bio-mass.

In a more preferred embodiment said inductor has an azo anthraquinonicor a stilbenic dye structure. In another more preferred embodiment saidinductor has a phenolic, aromatic or metallic structure.

In a preferred embodiment oxygen is added during the incubation of saidwhite-rot fungi in said pretreated liquid waste.

In another preferred embodiment a suitable amount of nutrients are addedduring the incubation of said white-rot fungi in said pretreated liquidwaste, to encourage the regeneration of cell activity of said white-rotfungi.

In a more preferred embodiment the nutrients are added in an amountranging from 0.5 to 4 percent in weight per volume.

In a preferred embodiment the pH during the incubation of said white-rotfungi in said pretreated liquid waste is ranging from 4 to 9 and thetemperature of incubation is ranging from 20 to 45° C.

In a more preferred embodiment the pH during the incubation of saidactive agents in said pretreated liquid waste is ranging from 2 to 7 andthe temperature of incubation is ranging from 20 to 70° C.

In another embodiment the pH during the incubation of said white-rotfungi or active agents thereof in said pretreated liquid waste isranging from 1 to 9 and the temperature of incubation is ranging from 18to 70° C.

In a preferred embodiment said white-rot fungi or active agents thereof,are incubated with said pretreated liquid waste for 2 hours to 14 days.

In another preferred embodiment said white-rot fungi or active agentsthereof, are incubated with said pretreated liquid waste for 20 minutesto 14 days.

In another embodiment, said fungi or active agents thereof obtainableafter step b) of the process are separated.

In another embodiment, the present invention relates to a process,wherein said separated fungi or active agents thereof are reused in theprocess for treating liquid waste.

In another embodiment, the present invention also relates to the use offungi or active agents thereof obtainable after step b) of the processfor treating liquid waste into a green waste composting process.

Further, the present invention provides a method for immobilising fungalactive agents on a support comprising the steps of:

-   -   culturing a fungus in a medium,    -   immersing a support with the supernatant of said fungal culture,        and    -   immobilising the active agents of said fungi on said support,        said agents being released in the fungal culture supernatant.

The aspects of this invention and other embodiments are more fully setforth in the following detailed description and the accompanying figuresand examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of white-rot fungi on the decoloration of NY3dye, with and without a pre-culture step expressed as a change ofabsorbance as a function of time.

FIG. 2 shows the effect of oxygen addition on the decoloration of NY3dye by white-rot fungi, expressed as a change of absorbance as afunction of time.

FIG. 3 shows the effect of a fungus immobilized on a support on thedecoloration of NY3 dye, expressed as a change of absorbance as afunction of time.

FIG. 4 shows the effect of different immobilization supports on effluentdecolorization during 8 or 20 days of fungal culture, with ozonolysis aspretreatment.

FIG. 5 represents a wavelength scan of the anthraquinonic dye NY3 duringits bio-transformation by white-rot fungi.

FIG. 6 shows the effect of adding different concentrations of maltsolution on the decoloration of NY3 dye by white-rot fungi, expressed asa change of absorbance as a function of time.

FIG. 7 shows the decoloration results obtained after treating crude orozonized-pretreated effluents by white-rot fungi (20 days of culture).

FIG. 8 shows the effect of white-rot fungi treatment on the toxicity ofcrude or ozonized effluents before or after said treatment.

FIG. 9 shows the mutagenicity of crude effluent, expressed as a ratiorec/pr1 as a function of the concentration of said effluent.

FIG. 10 shows the mutagenicity of ozonised effluent expressed as a ratiorec/pr1 as a function of the concentration of said effluent.

FIG. 11 shows the mutagenicity of crude effluent treated with white-rotfungi, expressed as a ratio rec/pr1 as a function of the concentrationof said effluent.

FIG. 12 shows the mutagenicity of ozonised effluent treated withwhite-rot fungi, expressed as a ratio rec/pr1 as a function of theconcentration of said effluent.

FIG. 13 shows the decoiorization results obtained after treating crudeor wood adsorption-pretreated effluents by white-rot fungi (8 days ofculture).

FIG. 14 shows the effect of laccases treatment on the decolorization ofcrude and ozonised effluents.

FIG. 15 shows the spectral change during decolorization of NY3 byconcentrated laccase added as a solution or after extraction throughadsorption on wood.

DETAILED DESCRIPTION

Several problems are currently observed with respect to the decolorationof dyes of effluents by means of fungi, which restrict the use of thefungi in industrial processes and applications.

One problem consists of the fact that the effluents inhibit fungalgrowth. As a consequence, the time required for efficient decolorationof effluents is extremely long. For this reason, the fermentation tanksfor use in such processes require non-realistic dimensions and suchprocesses are not economically interesting. Another problem consists ofthe fact that the media used for the fungal growth is not always usableat industrial scale. Another problem consists of the fact that up to nowthe mean criterion used to determine the efficiency of the fungalprocess is decoloration. Nevertheless some bacteria were demonstrated asforming toxic as well as carcinogenic compounds after degradation ofazoïc dyes. This could lead to a paradoxical situation in whichwastewater treated and uncolored could be thrown into the environmentwhile being more toxic than before treatment. The present inventionprovides a solution to these and other problems, by providing a methodfor decoloration and detoxification of liquid waste using fungi.

According to a preferred embodiment, the present invention relates to aprocess for treating liquid waste such as liquid waste from dyeindustries, compost effluent or agricultural effluent.

Said process permits the treatment of dyes-polluted liquid waste,wherein the dyes can be textile dyes, acid dyes, basic dyes, directdyes, reactive dyes, disperse dyes, and mixtures thereof. In a morepreferred embodiment said liquid waste is contaminated with azo dyes,anthraquinones dyes and/or stilbenic dyes.

In yet another more preferred embodiment said liquid waste iscontaminated with humic acids.

The first step of said process consists of a pretreatment step of theliquid waste. Said pretreatment may be physical and/or chemical,according to processes known in the art. In a preferred embodiment, saidpretreatment is selected from the group consisting of ozone treatment,adsorption process, including adsorption of the waste on biodegradablesupports, membrane filtration such as micro or nanofiltration, osmoseion exchange, electrolysis process, sodium borohydride process,electrochemical treatments such as cathodic and anodic process, directand indirect electrochemical oxidation, electrochemical in situsynthesis of oxidizing agents, electrodialysis, electromembraneprocesses, and electrochemical pre-oxidation, electrochemical ionexchange, electroflocculation, photochemical degradation, chemicaldegradation, Fenton's oxidation process.

According to a more preferred embodiment, said pretreatment is an ozonetreatment. As used herein “ozon treatment” is also referred to as“ozonolysis” or “ozonisation”.

The first pretreatment step may be performed for 10 minutes to 72 hoursaccording to the process used. More preferably, first pretreatment stepmay be performed for 0.5 to 10 hours according to the process used. Whenthe pretreatment is an ozone treatment, said liquid waste is preferablypretreated for 1 to 3 hours and more preferably for 1.5 hour ofdecoloration.

According to another more preferred embodiment, said pretreatment is anadsorption process, and preferably comprises adsorption of the waste ona biodegradable support, preferably a polymer support. The most commonadsorption supports are activated charcoal, silica gel, bauxite, peat,wood, cellulose derivatives or ions exchange resin. In a most preferredembodiment of this invention, the adsorption support used aspretreatment is comprised of wood shavings. Such wood shavings can becollected as an industrial by-product for example in saw-mills and infurniture industry. Advantageously such wood shavings are inexpensive.Suitable supports can be constituted of different woods including butnot limited to Betula sp., Fagus sp., Quercus sp. Pinus sp., Picea sp.Acer sp. Tilia sp., Populus sp., Castanea sp., Fraxinus sp., Juglanssp., Platanus sp. Teck sp., Meranti sp. or Cocos sp. The wood shavingsare dimensioned from sawdust to big wood shavings.

Preferably, said wood shavings are added to liquid waste to be treatedin the range of 0.1 to 100 g per liter. The time required for thetreatment of the waste can vary between 10 minutes and 3 days.

This first step allows a sensible decoloration of said liquid waste,from 150000 color units (APHA) to 80000 APHA in the case of ozonepretreatment and to 75000 APHA in the case of adsorption on a woodsupport. Said first step also permits a diminution on average of 10% ofthe chemical oxygen demand (COD).

The second step of said process consists of the treatment of saidpretreated liquid with fungi or active agents thereof. According to apreferred embodiment the fungi are white-rot fungi.

The white-rot fungi can be selected from the group comprising the genusAcanthophysium, the genus Aleurobotrys, the genus Aleurodiscus, thegenus Amphinema, the genus Amylostereum, the genus Armillaria, the genusAspergillus, the genus Asterostroma, the genus Auricularia, the genusBotryobasidium, the genus Botryohypochnus, the genus Calocera, the genusChaetomium, the genus Cladorrhinum, the genus Clitocybula, the genusColumnocystis, the genus Coriolopsis, the genus Cystostereum, the genusDaedalea, the genus Daedaleopsis, the genus Dichomitus, the genusDichostereum, the genus Echinodontium, the genus Fibulomyces, the genusFomitopsis, the genus Fusarium, the genus Ganoderma, the genus Grifola,the genus Hapalopilus, the genus Humicola, the genus Hymenochaete, thegenus Hyphoderma, the genus Hyphodontia, the genus Hypochnicium, thegenus Inonotus, the genus Irpex, the genus Laurilia, the genusLaxitextum, the genus Lentinus, the genus Lenzites, the genus Lentinula,the genus Leucogyrophana, the genus Lycoperdon, the genus Marasmius, thegenus Merulius, the genus Mycoacia, the genus Myrothecium, the genusPaecilomyces the genus Panellus, the genus Penicillium, the genusPeniophora, the genus Perenniporia, the genus Pestalotia, the genusPhanerochaete, the genus Phellinus, the genus Phlebia, the genusPholiota, the genus Pleurotus, the genus Polyporus, the genus Poria, thegenus Punctularia, the genus Pycnoporus, the genus Resinicium, the genusSchizophyllum, the genus Scytinostroma, the genus Steccherinum, thegenus Trametes, the genus Trichoderma, the genus Tyromyces and the genusVararia. Table 1 in example 3, lists several white-rot fungi strains,which have been successfully used, according to said process.

According to a preferred embodiment, said white-rot fungi belong to thegenus Acantophysium, the genus Chaetomium, the genus Clitocybula, thegenus Coriolopsis, the genus Cystostereum, the genus Ganoderma, thegenus Lentinus, the genus Lentinula, the genus Lenzites, the genusMerulius, the genus Paecilomyces, the genus Perenniporia, the genusPhanerochaete, the genus Phellinus, the genus Phlebia, the genusPolyporus, the genus Pycnoporus and the genus Trametes. More preferablysaid white-rot fungi belong to the genus Acanthophysium, the genusCoriolopsis, the genus Clitocybula, the genus Cystostereum, the genusGanoderma, the genus Paecilomyces, the genus Perenniporia, the genusPhellinus, the genus Phlebia, the genus Pycnoporus and the genusTrametes.

According to a more preferred embodiment, said white-rot fungi areselected form the group consisting of Acantophysium bisporum MUCL 32213,Coriolopsis polyzona MUCL 38443, Cystostereum murraii MUCL 33747,Ganoderma subamboinense MUCL 38859, Lentinus cladopus MUCL 28678,Lentinula edodes MUCL 29756, Lenzites betulina MUCL 38559, Meruliustremelosus MUCL 38065, Paecilomyces variotii MUCL 21705, Perenniporiamedulla-panis MUCL 40050, Perenniporia ochroleuca MUCL 41114,Perenniporia tephropora MUCL 41562, Phanerochaete chrysosporium MUCL19343, Phanerochaete ericina MUCL 33845, Phellinus rimosus MUCL 38446,Phlebia subserialis MUCL 33724, Polyporus brumalis MUCL 29280, Polyporusciliatus MUCL 40141, Pycnoporus cinnabarinus MUCL 38520, Pycnoporuscoccineus MUCL 38525, Pycnoporus sanguineus MUCL 41625 and MUCL 41582,Trametes versicolor MUCL 38412 and MUCL 28407. Yet more preferably saidwhite-rot fungi are selected from the group consisting of Acantophysiumbisporum MUCL 32213, Coriolopsis polyzona MUCL 38443, Cystostereummurraii MUCL 33747, Paecilomyces variotii MUCL 21705, Perenniporiamedulla-panis MUCL 40050, Perenniporia ochroleuca MUCL 41114,Perenniporia tephropora MUCL 41562, Phellinus rimosus MUCL 38446,Phlebia subserialis MUCL 33724, Pycnoporus coccineus MUCL 38525,Pycnoporus sanguineus MUCL 41625 and Trametes versicolor MUCL 38412.

According to another more preferred embodiment, said white-rot fungi areselected from the group consisting of Clitocybula dusenii DSM 11238,Trichoderma harzanium MUCL 29707 and Trichoderma longibrachiatum MUCL39887.

According to the invention the white-rot fungi are cultured usingtechniques known in the art. These fungi are preferably grown in richmedia comprising a carbon source, a nitrogen source and mineral salts.Said white-rot fungi can also be grown on wood such as wood shavings,which constitute an excellent source of nutrients. They can be incubatedat temperatures ranging from 20 to 45° C. and at a pH ranging from 4 to9, under shaking conditions or not. According to a preferred embodiment,said white-rot fungi are grown in a medium containing malt extract in aconcentration ranging from 0.5 to 8% (w/v). In a more preferredembodiment said, white-rot fungi are grown in a 2% malt medium.

During said second step, active agents isolated from said white-rotfungi can also be used to treat said pretreated liquid waste. The term“active agents” as used herein, encompass white-rot fungi crude cellextracts, semi-purified cell extracts, concentrated cell extracts,isolated or purified agents, such as enzymes or a mixture of enzymes.According to a preferred embodiment said active agents are enzymes suchas tyrosinase, hydrolytic enzymes such as hydrolases, cellulolyticenzymes such as cellulase or xylanases, or ligninolytic enzymes such asextracellular oxidases and peroxidases, lacasses, lignin- or Mnperoxidases, cellobiose deshydrogenase, produced by said white-rotfungi. In another preferred embodiment, the white-rot fungi activeagents comprise hydrolytic, cellulolytic or ligninolytic enzymes. Morepreferably said active agents are selected from the group consisting oflaccases, lignin peroxidases and manganese peroxidases, or mixturesthereof. According to a yet more preferred embodiment said active agentsare laccase enzymes. In yet another preferred embodiment, said activeagents may also comprise small molecules such as organic acids, aromaticactive compounds or mixture thereof.

In another preferred embodiment, the white-rot fungi active agents areemployed as raw preparation, as purified enzymes, or in an immobilizedform. Preferably, according to yet another more preferred embodiment,the white-rot fungi active agents are immobilized on a wood support,e.g. on wood shavings.

During said second step, said white-rot fungi may be added to saidpretreated liquid waste as a pure, mixed, or enriched culture as cells,mycelium fragments, spores, pre-culture inoculum, culture broth, or assupernatant. According to another embodiment, said white-rot fungi canbe added to said pretreated liquid, either singly, or in combinationwith other microorganisms such as fungi or bacteria.

According to yet another embodiment, the white-rot fungi to be added tosaid pretreated liquid waste may be encapsulated in a matrix consistingof polymers. In a preferred embodiment said polymers consist ofbiodegradable, natural, non-toxic polymers. More preferably saidpolymers are selected from the group consisting of alginate salts,kappa-carrageenan salts, iota-carrageenan salts, maltodextrin, wheyprotein concentrate (WPC), skimmed milk powder (SMP), dried yeastautolysate (YA), dried yeast extract (YE), corn starch (CS), modifiedstarch (MS), and polyvinylalcohol. Yet more preferably said polymers areselected from the group consisting of alginate salts, kappa-carrageenansalts and iota-carrageenan salts. Other suitable polymers comprisecellulose or polypropylene. In another embodiment, also the activeagents of said fungi to be added to said pretreated liquid waste may beencapsulated in a matrix consisting of polymers. Said encapsulated formof the fungi or the active agents thereof is also meant for conservationof the fungi or the active agents thereof and can be inoculated inpre-culture. Thus, in another preferred embodiment said white-rot fungior the active agents thereof are conserved and inoculated in pre-culturein an encapsulated form, i.e. in a matrix consisting of polymers.

According to another embodiment, during said second step, said white-rotfungi could be further immobilized. More preferably, said white-rotfungi are immobilized on a support such as stainless steel support orsupport made of bio-beads such as those usually used for the biologicalcleaning of fish tanks, for example DUPLA Biokaskade or Minikaskadebio-beads. These bio-beads are usually made of polymer such aspolypropylene. Said white-rot fungi can also be immobilized on wood,more particularly on wood shavings. Said white-rot fungi can also beimmobilized on air filters, e.g. on Vileda Dunstfilters. Examples ofsuitable matrices include but are not limited to stainless steel, orsynthetic polymer such as polypropylene. According to a preferredembodiment, said white-rot fungi are preferably immobilized on astainless steel support, which is preferably in a mesh, a web form, oran interwoven or entangled mass of stainless steel strands.

The white-rot fungi active agents, such as the laccases for example, mayalso be used in immobilized form using the immobilization techniquesmentioned below.

According to another embodiment, in order to improve the efficiency ofthe white-rot fungi treatment of said pretreated liquid waste, it isalso possible to add an inductor to the white-rot fungi culture mediapreferably after said fungi has reached a significant bio-mass. Theinductor will preferably have an azo, an anthraquinonic or a stilbeniclike structure. Non limiting example of inductors are shown in example4. Other inductors such as ferulic acid or xylidine can also be used. Inanother more preferred embodiment said inductor has a phenolic, aromaticor metallic structure. When the inductor is added at the beginning ofthe fungus growth, the bio-transformation of the dye happens but alatent period is observed.

During the second step, the white-rot fungi or active agents thereof areincubated with said pretreated liquid waste, which can be provided in acontinual or sequential way and may be collected at the end of thetreatment also in a continual of sequential way. According to apreferred embodiment, said pretreated liquid waste is first diluted from2 to 10 times before treatment with white-rot fungi or active agentsthereof. Alternatively, said pretreated liquid waste can also be usedundiluted, preferably when the liquid waste is to be treated with activeagents.

According to another embodiment, oxygen may be added during the secondstep treatment i.e. during the incubation of said white-rot fungi insaid pretreated liquid waste. Said oxygen addition will further improvethe process's efficiency, by improving the development and the survivalrate of said white-rot fungi. Moreover, the oxygen plays a major role inthe oxidative fungal ligninolytic mechanisms, which is directly involvedin the degradation of various compounds including dyes.

According to yet another embodiment, a suitable amount of nutrients canfurther be added during the second step i.e. during the incubation ofsaid white-rot fungi in said pretreated liquid waste. This nutrientaddition will improve the regeneration of cell activity of saidwhite-rot fungi. Non limiting examples of nutrients are malt extract,beetroot pulp residues, molasses, bagasses and other nutrient sourcescontaining sugars. In a preferred embodiment, the nutrients are added inan amount ranging from 0.5 to 4 percent (w/v).

According to a preferred embodiment, the treatment of said pretreatedliquid waste with said white-rot fungi, can be performed at a pH rangingfrom 4 to 9 and at a temperature ranging from 20 to 45° C. Theincubation time is preferably ranging from 3 to 14 days.

According to another preferred embodiment, the treatment of saidpretreated liquid waste with said white-rot fungi active agents, can beperformed at a pH ranging from 2 to 7 and at a temperature ranging from20 to 70° C. The incubation time will preferably range from 2 to 24hours.

In a more preferred embodiment the pH during the incubation of saidwhite-rot fungi or active agents thereof in said pretreated liquid wasteis ranging from 1 to 9 and the temperature of incubation is ranging from18 to 70° C. In another preferred embodiment said white-rot fungi oractive agents thereof, are incubated with said pretreated liquid wastefor 20 minutes to 14 days.

The present invention therefore relates to a process wherein saidpretreatment in combination with a treatment with white-rot fungi oractive agents thereof are effective in removing from 28.4 percent to99.5 percent of the color of said liquid waste as measured by the changein optical density before and after pretreatment and incubation withsaid white-rot fungi or active agents thereof.

The present invention also relates to a process, wherein saidpretreatment in combination with a treatment with white-rot fungi oractive agents thereof are effective in detoxifying said liquid waste asmeasured by the toxicity of said liquid waste on human cells, before andafter pretreatment and incubation with said white-rot fungi.

In another embodiment, said fungi or active agents thereof obtainableafter step b) of the process are separated. In another embodiment, thepresent invention relates to a process, wherein said separated fungi oractive agents thereof are reused in the process for treating liquidwaste.

In another embodiment, the present invention also relates to the use offungi or active agents thereof obtainable after step b) of the processfor treating liquid waste into a green waste composting process. Ingeneral, a green waste composting process is generally very slow becauseof the presence of quantities of low degradable lignocelluloticresidues, phenolic compounds and humic acids. The fungi that are used inthe process for treating liquid waste can be further valorized in greenwaste composting processes. These fungi, since they produce lignolyticenzymes, enable to improve the green waste composting process. They canbe added in the primarily phase of the composting to initiate thedegradation of compounds and facilitate the following bacterialtransformations. In this case fungal biomass will be killed during thethermophilic phase of composting and presents no risk for theenvironment. They can also be used in the final maturation step ofcomposting, but in this case, only non-pathogenic fungal strains can beused. Strains comprised herein here have been selected to be GRAS(generally recognized as safe). An example of non-pathogenic strain isPycnoporus sanguineus MUCL 41582.

In another preferred embodiment, fungal biomass which has been grown onwood chips as immobilization support is transferred after watertreatment into a green waste composting process to improve this latterprocess. The wood chips have the advantage over the other supports to bebiodegradable and the fungal biomass produced on the wooden support canbe directly transferred into green waste composting plants, without theneed for a complicated step to separate the biomass from the support.

The present invention provides an easy and inexpensive method forimmobilizing said active agents on a support. Further, the use of theactive agents, preferably enzymes, in an immobilized form enables toobtain said enzymes in a concentrated form, without having to resort toother enzyme concentration techniques such as dialysis, ultrafiltrationor the use of columns. The method for immobilising fungi active agentson a support comprises the steps of:

-   -   culturing a fungus in a medium,    -   immersing a support with the supernatant of said fungal culture,        and    -   immobilising the active agents of said fungi on said support,        said agents being released in the fungal culture supernatant.

In a preferred embodiment, said method comprises the immobilisation offungi active agents on a wood support, and consists of preparing afungal culture, immersion of wood shavings or sawdust into theextracellular fluids of the fungal culture and immobilizing the enzymesreleased into the extracellular fluids of the fungal culture on the woodshavings. According to a yet more preferred embodiment said wood can beadded to the culture supernatant in quantity comprised between 0.1 and100 g per liter of growth medium, during 1 second to 24 hours.

The present invention describes a process, which is sustainable. As usedherein, the term “sustainable” relates to the fact that the fungalbiomass used in the process for treatment of liquid waste can be re-usedfor the same process of liquid waste treatment, but may be also furtherre-used in other applications, such as for the improvement of greenwaste composting processes. Furthermore, the process is environmentalfriendly.

The present invention will be further described hereunder by way ofnon-limiting examples.

EXAMPLES Example 1

In a preferred embodiment of the process according to the invention, thepretreatment step in said process is an ozonisation.

The ozone is produced from pure oxygen using an ozone generator of typeOZONIA ZF10AT which can produce 10 to 30 kg of ozone per hour and cantreat 800 m³ of industrial liquid waste per day. The concentration inozone produced is 10% w/w (O₃/O₂), the quality is controlled byspectrophotometry at 258 nm by passing the gas phase through a flowcell. The ozonisation of liquid waste is performed in a 50 m³ verticalreaction tank. A radial diffuser allows the injection of the gas in saidliquid waste through a venturi tube. The flow rate of the gas variesfrom 11 to 22 m³ per hour. The liquid waste to be treated by ozonisationis usually at pH 5 (±0.5) and at a temperature of 30 to 35° C. The idealincubation time is 1.5 hours, which is obtained by recycling said liquidwaste through the tank. This ozone treatment allows a decrease of thecolor in said liquid waste from 150000 color units (APHA) to 80000 colorunits (APHA) and also a diminution of the COD on average of 10%.

APHA is an abbreviation for “American Public Health Association”, whichpublishes a collection of test procedures for water and wastewater. Itdescribes the determination of the color standard in Hazen units,wherein 1 mg/l Pt equals one Hazen. The stock solution is prepared asfollows: Dissolve 2.49 g K₂PtCl₆ and 2.02 g CoCl₂.6H₂O in 200 ml ofconcentrated pure HCl (d=1.19) and dilute to 1 liter with distilledwater. The absorbance of this solution at 455 nm represents 1000 Hazen.This method was elaborated to measure the color of surface water, whichgenerally absorb light in this region (455 nm). In the case of coloredwastewater, we use a spectrophotometric integrative method (SIM), andthe results are converted in color units (APHA). This method is able tomeasure coloration of the water in the visible spectrum. In practice,the absorbance of the sample is measured in the visible region (from 380to 740 nm). The integration of the curve absorbance/wavelength gives anumerical result in area units. To convert this result in color units(APHA), 5 dilutions of the stock solution of the standard (containingPt) described above are measured by the two methods Hazen and SIM. Agraph area/hazen units is constructed for the standard. A conversionfactor is calculated, and the result obtained for the sample can beconverted through the conversion factor and expressed in color units(APHA).

The chemical oxygen demand (COD) is the amount of oxygen required tooxidize by chemical means organic carbon compounds completely to CO₂ andH₂O. In practice, organic matter in water is oxidized by K2Cr2O7 underrather stringent conditions. The amount of dichromate oxygen used isdetermined and expressed as COD. The method used is a normalized method(NFT 90.101 or DIN 38 409-H41-1) based on a photometric determination ofchromium (III) concentration after 2 hours of oxidation with potassiumdichromate/sulfuric acid/silver sulfate at 148° C. A 620 nm filter isused.

Example 2

A non-exhaustive list of suitable pretreatment according to theinvention is given hereunder.

Adsorption:

The most commons adsorption supports are activated charcoal, silica gel,bauxite, peat, wood, cellulose derivatives, ions exchange resin.

For illustrating the pretreatment of adsorption on wood, the followingexample is given. In this example, wood shavings are constituted bypieces of about 4 square centimeters from Fagus sylvatica, obtained as aby-product from a furniture industry. Said wood shaving are added in aconcentration of 3 g per liter and incubated during 24 hours. The colorof a dye industry effluent was measured before and after thispretreatment and a decrease from initial 105 000 to 72 000 color units(APHA) was measured. This result is nearly the same as obtained throughozonolysis, but the costs related to this latter method are considerablyreduced.

Membrane Filtration:

Ultrafiltration, micro or nanofiltration: use a combination of activatedcarbon adsorption followed by a membrane separation (Sandoz). Theactivated charcoal adsorbs the most resistant elements which have a lowmolecular weight and the membranes such as nanofiltration membranes stopthe elements with a molecular weight higher than 1000.

Electrolysis:

Electrolysis is a process, which was initially reserved to liquidcontaminates with metals, however, this process permits a decrease ofthe coloration of liquid waste of about 90 to 95%.

Process Using Sodium Borohydride (Type Reading, UK):

This process consists of first adjusting the pH of the liquid waste topH 5.5 and subsequently adding to said liquid waste, successively, asolution of sulfuric acid, a flocculent (Metafloc 137), a solution ofsodium bisulfate, a solution of sodium borohydride in a basic solution(Morton). This process allows the decrease of the COD on average of 33%and eliminates 85% of the amount of copper and decreases the color byabout 90%.

Electrochemical Treatments:

In electrochemical treatments, oxidation is achieved by mean ofelectrodes, where a determined difference of potential is applied,dipped in the effluent to treat. Efficiency of the method is a functionof several parameters difference of potential, nature of the electrodes,pH. On this principle, several different processes have been developedas cathodic and anodic processes, direct and indirect electrochemicaloxidation, electrochemical in situ synthesis of oxidizing agents,electrodialysis, electromembrane processes, and electrochemical ionexchange.

Electroflocculation:

Electroflocculation is the combination of an oxidation, a flocculationand a flotation and involves the electrolytic addition of coagulatingmetal ions directly from sacrificial electrodes.

These ions coagulate with pollutants in the water, in a similar mannerto the addition of coagulating chemicals such as aluminum chloride andferric chloride, and allow the easier removal of the pollutants. Theprocess involves the application of an electric current to sacrificialelectrodes, usually aluminum, inside a processing tank. The reactions atthe anode and cathode respectively are typically generating aluminumions as a coagulating agent as well as gas bubbles. The well-knownproperties of the aluminum ions as a coagulating agent cause them tocombine with the pollutants. The gas bubbles generated can capture thecoagulated agglomerates, resulting in most of the pollutant beingfloated to the surface.

Photochemical Degradation:

Permits the transformation of E-isomers to Z-isomers using UV-Visirradiation.

Chemical Degradation:

Usually using oxidizing agents. The most common oxidizing agents arehypochlorite, chlorine gas chlorine dioxide, hydrogen peroxide, ozone,and potassium permanganate. Chlorine based oxidizing agents areparticularly efficient on monoazo dyes and anionic anthraquinonic dyes.

Fenton's Oxidation Process:

In the presence of a catalyst, a hydrogen peroxide solution formshydroxyl radicals (OH) of strong oxidizing power or nascent oxygen (O).This hydroxyl radical, having powerful oxidizing power, can oxidize mostorganic substances including dechlorinating organic chlorine compounds.In liquid waste treatment, organic substances are decomposed by mixingwaste water with hydrogen peroxide and iron catalyst. Then liquid wasteis neutralized. This process can decompose bio-persistent coloredsubstances.

Example 3

A non-exhaustive list of fungi tested during the process according tothe invention is shown in table 1. These fungi are deposited in thefungal library MUCL (Mycotheque de l'Universite catholique de Louvain).TABLE 1 Genus Species N. MUCL Acanthophysium bisporum 32213Acanthophysium cerussatum 32645 Acanthophysium lividocaeruleum 33688Aleurobotrys botryosus 32323 Aleurodiscus aurantius 33921 Aleurodiscusgabonicus 32433 Aleurodiscus wakefieldiae 34807 Amphinema byssoides32977 Amylostereum areolatum 32874 Amylostereum chailletti 32912Amylostereum laevigatum 33857 Armillaria gallica 31339 Aspergillus niger19001 Asterostroma cervicolor 38354 Asterostroma laxum 38356Asterostroma ochroleucum 38358 Auricularia auricula 28689 Auriculariaauricula 38073 Auricularia cornea 28966 Auricularia fuscosuccinea 28965Auricularia polytricha 30975 Auricularia polytricha 38067 Botryobasidiumcandicans 33808 Botryobasidium sphaericosporum 32749 Botryobasidiumsphaericosporum 32750 Botryohypochnus isabellinus 33809 Calocera viscosa31690 Chaetomium brasiliense 19261 Chaetomium globosum 9597 Chaetomiumglobosum var. griseum 39527 Chaetomium pachypodioides 9586 Cladorrhinumfoecundissimum 4060 Clitocybula dusenii DSM 11238 Collybia peronata20939 Collybia reinakeana 38064 Columnocystis abietina 33928 Coriolopsispolyzona 38443 Corticium meridioroseum 34729 Cystostereum murraïi 33747Daedalea quercina 11661 Daedalea quercina 30382 Daedaleopsis confragosa29566 Dendrophora albobadia 33710 Dichomitus leucoplacus 41472Dichostereum durum 32558 Dichostereum effuscatum 33642 Dichostereumgranulosum 33644 Dichostereum peniophoroides 32336 Dichostereumsordulentum 32712 Dichostereum effuscatum 32221 Dichostereum orientale32644 Dichostereum pallescens 32640 Dichostereum ramulosum 32279Dichostereum rhodosporum 32191 Dichostereum sordulentum 32167Echinodontium tinctorium 1005 Fibulomyces septentrionalis 34891Fomitopsis rosea 40102 Fusarium annulatum 8059 Fusarium concolor 797Fusarium incarnatum 38815 Fusarium oxysporum 1064 Fusarium reticulatum19032 Ganoderma carnosum 39430 Ganoderma dejongii 39643 Ganodermaoerstedii 38857 Ganoderma subamboinense 38859 Grifola frondosa 31544Hapalopilus rutilans 28390 Humicola brunnea 8355 Humicola fuscoatra 8799Humicola nigrescens 14437 Humicola nigrescens 21866 Humicola parvispora19494 Humicola nigrescens 7913 Hymenochaete boidinii 32028 Hymenochaetecruenta 38613 Hymenochaete rubiginosa 31546 Hymenochaete separata 32762Hymenochaete tabacina 28221 Hymenochaete pinnatifida 32735 Hyphodermadeviatum 32102 Hyphoderma litschaueri 33820 Hyphoderma mutatum 32950Hyphoderma puberum 38042 Hyphoderma setigerum 38043 Hyphodermavariolosum 32509 Hyphodontia alutaria 34734 Hypochnicium vellereum 34735Hypochnicium eichleri 32105 Hypochnicium punctulatum 33699 Inonotushispidus 35148 Inonotus leporinus 40107 Irpex vellereus 32181 Irpexlacteus 31500 Laurilia sulcata 40113 Laxitextum bicolor 32182 Laxitextumbicolor 33705 Lentinula edodes 29756 Lentinus boryanus 30973 Lentinuscladopus 28678 Lentinus lepideus 40109 Lentinus tigrinus 28826 Lenzitesbetulina 38552 Lenzites betulina 38559 Leucogyrophana pinastri 30922Leucogyrophana pinastri 34739 Leucogyrophana pinastri 39391 Lycoperdonfoetidum 28389 Marasmius androsaceus 31691 Marasmius androsaceus 35155Marasmius oreades 28591 Merulius tremellosus 38065 Mycoacia nothofagi33992 Myrothecium verrucaria 19018 Paecilomyces cremeoroseus 9652Paecilomyces marquandii 4138 Paecilomyces inflatus 8231 Paecilomycesinflatus 34987 Paecilomyces variotii 21705 Paecilomyces variotii 28553Paecilomyces variotii 28975 Paecilomyces variotii 30859 Paecilomycesvictoriae 9651 Paecilomyces marquandii 18884 Panellus serotinus 31030Penicillium expansum 38789 Peniophora aurantiaca 33768 Peniophorafissilis 32709 Peniophora gigantea 1001 Peniophora incarnata 30546Peniophora lycii 35220 Peniophora piceae 31774 Peniophora polygonia32112 Peniophora proxima 33999 Perenniporia ellisiana 39555 Perenniporiaformosana 38828 Perenniporia fraxinea 35180 Perenniporia fraxinea 39556Perenniporia fraxinea 41509 Perenniporia fraxinophila 39561 Perenniporiafraxinophila 39822 Perenniporia maackiae 38881 Perenniporia martius40486 Perenniporia medulla-panis 40050 Perenniporia medulla-panis 38746Perenniporia narymica 39551 Perenniporia ochroleuca 39819 Perenniporiaochroleuca 41114 Perenniporia ohiensis 38827 Perenniporia ohiensis 39727Perenniporia podocarpi 40483 Perenniporia subacida 39553 Perenniporiasubacida 39820 Perenniporia tephropora 41562 Pestalotia oxyanthi 35070Pestalotia populi-nigrae 31398 Pestalotia subcuticuralis 7966 Pestalotiasubsessilis 38335 Phanerochaete chrysosporium 19343 PhanerochaeteChrysosporum 31762 Phanerochaete chrysosporium 38489 Phanerochaeteravenelii 33671 Phanerochaete salmonicolor 30464 Phanerochaete sordida34000 Phanerochaete sanguinea 30740 Phanerochaete tamariciphila 39084Phanerochaete tuberculata 33892 Phanerochaete ericina 33845 Phellinusferreus 28239 Phellinus hartigii 31400 Phellinus jezoënsis 38884Phellinus laevigatus 1006 Phellinus rimosus 38446 Phellinus tuberculosus35098 Phellinus alni 38882 Phellinus contiguus 30799 Phellinus rimosus38446 Phlebia livida 33615 Phlebia ludoviciana 34701 Phlebia radiata30503 Phlebia radiata 39535 Phlebia subcalcea 32243 Phlebia subochraceus33903 Phlebia subserialis 33724 Pholiota adiposa 7900 Pholiota lenta28253 Pholiota nameko 31614 Pleurotus calyptratus 28909 Pleurotuscolumbinus 38096 Pleurotus cornupiae 31683 Pleurotus eous 38684Pleurotus eryngii 31538 Pleurotus flabellatus 38085 Pleurotus floridanus38055 Pleurotus pulmonarius 34667 Pleurotus sajor-caju 38076 Polyporusumbellatus 31707 Polyporus arcularius 40124 Polyporus brumalis 40131Polyporus ciliatus 30563 Polyporus grammocephalus 39575 Polyporusmeridionalis 40149 Polyporus dictyopus 40147 Polyporus tenuiculus 40151Polyporus tubaeformis 39667 Polyporus tuberaster 39586 Polyporusbrumalis 29280 Polyporus ciliatus 40141 Poria placenta 30853 Punctulariatuberculosa 33849 Pycnoporus cinnabarinus 38518 Pycnoporus cinnabarinus38520 Pycnoporus cinnabarinus 38607 Pycnoporus cinnabarinus 38620Pycnoporus coccineus 38524 Pycnoporus coccineus 38525 Pycnoporuscoccineus 38527 Pycnoporus sanguineus 28499 Pycnoporus sanguineus 30513Pycnoporus sanguineus 38530 Pycnoporus sanguineus 38531 Pycnoporussanguineus 39259 Pycnoporus sanguineus 41582 Pycnoporus sanguineus 41594Pycnoporus sanguineus 41625 Pycnoporus sanguineus 41627 Pycnoporussanguineus 41660 Pycnoporus puniceus 41780 Resinicium bicolor 31716Schizophyllum commune 31016 Scytinostroma mediterraneense 34754Scytinostroma renisporum 32568 Steccherinum rhois 32827 Trametes gibbosa29020 Trametes zonatella 40172 Trametes varians 40171 Trametesversicolor 28407 Trametes versicolor 38412 Trichoderma longibrachiatum29753 Trichoderma harzianum 29707 Trichoderma longibrachiatum 39887Tyromyces kmetii 39628 Vararia breviphysa 32576

Example 4

Examples of chemical structures of inductors useful in said processaccording to the invention and their sterilization protocol are givenhereunder.

Prior to being added to the fungal culture, the inductors can besterilized by tyndallisation which consists of a sterilization processat low temperature, which has the advantage of preserving the inductorfrom being destroyed. The process consists of measuring the suitablequantity of inductor, adding the inductor to a glass tube, heating thetube for 1 hour at 60° C. in a water bath, then let is cool for 24hours, repeating the heating and cooling steps for 3 to 5 times.

In an example, the inductors NY3, NY5 and RBBR were added to a fungalculture of Pycnoporus sanguineus MUCL 41582 after 2 days of pre-culture.Laccase activity was measured after 5 days of incubation of the culturewith the inductors. Results are indicated in the below-given Table 2 andshow clearly that laccase activity is enhanced when the fungus iscultured in the presence of inductors. A more than 20 fold increase wasobserved in the case of the addition of the inductor NY5. TABLE 2Inductors Without inductor NY3 NY5 RBBR Enzymatic activity (nmol/ml)0.41 3.87 11.60 5.40

Example 5

Examples of media and inoculation procedures for the white-rot fungi aregiven hereunder.

Malt medium (2%) (ML2): White-rot fungi are best grown in a malt extractmedium, which consists of a solution of 2% (w/v) of malt extract inwater. The medium is sterilized by autoclaving for 18 min, at 120° C.,at a pressure of 1.2 bars.

Preparation of inoculum using mycelium fragments from a pre-culture ofthe fungus on a malt-agar media (2%). The medium is prepared with 2% w/vmalt extract and 1.5% w/v agar in water. The medium is sterilized byautoclaving for 15 min, at 120° C., at a pressure of 1.2 bars.

A pre-culture is performed by inoculating a fragment of the fungus on amalt extract-agar (2%) (MA2) plate. The pre-culture is incubated from 3to 10 days at 25° C. preferably. The fermenting tanks are typicallyinoculated with 150 fragments of pre-cultured fungi per litre ofculture, wherein each fragment corresponds to a 3 mm or 6 mm diameterpiece of fungi taken from the petri dish.

Preparation of inoculum using ground mycelium which is furtherencapsulated in a matrix: The matrix consists of polymers such asalginate salts, kappa-carrageenan salts and iota-carrageenan salts. Thepolymer solutions are prepared with 2.5 to 3% w/v of polymer in water.

The mycelium from a white-rot fungus is obtained from a pre-culture ofsaid white-rot fungus in MA2 medium as described above. Fragments fromthis pre-culture are added to liquid medium ML2 and are incubated for 7to 10 days at 25° C. under orbital shaking. When the mycelium presents apellet-like aspect; it is further ground during 15 seconds at 20 rpm.The ground mycelium is mixed to a solution of polymer in a 1:1 ratio(v/v), and the mixture is further extruded with CaCl₂ or KCl solution(0.1 M for alginates, 0.4 M for carrageenans) such as to form beads. Thefermenting tanks are typically inoculated with 150 beads per litre ofculture.

Example 6

The influence of the pre-culture of white-rot fungi on the decolorationprocess of the anthraquinonic dye NY3 using said white-rot fungi isdescribed hereunder.

During this experiment, a pre-culture of a white-rot fungus was firstperformed in ML2 medium as described above. NY3 dye was later added tothe fungal culture such as to obtain a concentration in NY3 of 0.7 g/l.The decoloration was measured spectrophotometrically. The resultsobtained are illustrated FIG. 1. The graph shows that the decolorationstarts faster when the fungus has a significant biomass before the dyeis added to the culture.

Example 7

This example provides evidence for the positive effect of the additionof oxygen to a culture of a white-rot fungus on the performance of thewhite-rot fungus to decolorize the anthraquinonic dye NY3.

In general, white-rot fungi are aerobic organisms. They require asufficient amount of oxygen to survive and to develop. Moreover, oxygenplays a major role in their oxidative fungal ligninolytic mechanism.

In this example the effects of an oxygen addition during culturing ofthe fungus on the bio-transformation of the dye by the white rot funguswas studied. The fungus Pycnoporus sanguineus MUCL 41582 was cultured inML2 medium to which the NY3 dye was added and the effects of an additionof oxygen on the bio-transformation of the dye were investigated. Oxygenwas added during approximately 15 minutes per day at the pressure of 0.2bars. The decoloration of the anthraquinonic dye NY3 by Pycnoporussanguineus was followed spectrophotometrically. FIG. 2 shows thefavorable effect of this addition of oxygen on the decolorization of NY3by white-rot fungi.

Example 8

This example illustrates the decoloration of dyes as well as effluentswhen using a fungus, which has been immobilized on a support. Differentsupports were tested.

In a first example the decoloration of the anthraquinonic dye NY3 withwhite-rot fungi immobilized on a stainless-steel support was studied.The presence of an immobilizing support in agitated liquid culture isgenerally beneficial for the growth, the development and theperformances of the fungus. In the setting of the follow-up ofdecoloration of dyes, the immobilization support should not interferewith the experiment; they must be neutral towards the dyes. Thedecoloration of the anthraquinonic dye NY3 by a fungal strain has beenfollowed spectrophotometrically and a comparison of the results has beenmade between the speed of decoloration with or without immobilization.The results shown in FIG. 3 demonstrate that immobilization of the fungion stainless-steel support plays an important role in the improvement ofthe decoloration of the liquid containing the NY3 dye.

In a second example the decoloration of an industrial dye effluent wasstudied. The pretreatment consisted of an ozonolysis. The second step inthe process for decoloration consisted of a treatment with a fungusimmobilized on a support. Different immobilization supports, i.e. a woodsupport, biobeads, a stainless steel support and hoodfilters, werecompared. The used fungus in this process was Pycnoporus sanguineus MUCL41582. The efficiency of the different supports was determined bymeasuring the decoloration of the industrial dye 8 days or 20 days aftertreatment with the immobilized Pycnoporus sanguineus MUCL 41582. Resultsare shown in FIG. 4.

After eight days wood chips were the most efficient support, because ina first phase the wood chips adsorb the dyes present in the effluent.These dyes were then transformed by the fungus and the wood chipsappeared uncolored at the end of transformation. For kinetic reasons,the transformation was enhanced as compared with the other supports,which are inert towards the dyes. Biobeads, hoodfilters and stainlesssteel showed approximately the same capabilities to immobilize thefungal biomass and to decolorize the effluent.

In another example, two other examples of effluents, 80949 and 80990,were treated by Perenniporia tephropora 41562 cultured on a wood chipssupport (no pretreatment). The effluent numbered 80949 is a textileeffluent that contains mainly basic dyes for acrylic dying; the effluentnumbered 80990 is a textile effluent that contains mainly dispersed dyesfor polyester dying. In addition, a compost lixiviate was treated byTrichoderma harzanium MUCL 39887 (no pretreatment). The compostconsisted of lixiviate from a composting society that was mainly coloredby humic acids. Results are presented in table 3. TABLE 3 Effluents80949 80990 Compost Decoloration 48% 20% 87%

Results showed that the fungus Perenniporia tephropora 41562 cultured ona wood chips support can decolorize effluents containing basic dyes anddispersed dyes. The effluents were decolorized to 48% and 20% for the80949 and the 80990 textile effluents respectively.

The compost lixiviate, treated with Trichoderma harzanium wasdecolorized up to 87%.

These results also show the efficiency of the fungus Trichodermaharzanium to degrade humic acids, which are present in the compostlixiviate, and therefore provide evidence for the interest of suchimmobilized fungal biomass for the improvement of green wastecomposting.

Example 9

In this example, the decoloration of the anthraquinonic dye NY3 usingwhite-rot fungi was studied. The effect of the addition of nutrients,i.e. a malt extract, as well as the adsorption of the dye on the biomasswas addressed.

Previous experiments (see also example 8, FIG. 3) showed the evolutionof the absorbance at 599 nm of the anthraquinonic dye NY3 during itsbio-transformation by a fungal strain (FIG. 3). The absorbance measuredat the beginning of the reaction, decreased quickly during the firsthours of reaction (see FIG. 3). The spectrophotometric follow-up (FIG.5) showed that a red intermediate presenting a peak at 500 nm was formedwhich then disappeared more slowly. The significant spectrum changesindicated that decoloration was caused by transformation of dyes ratherthan by a physical phenomenon of adsorption. This result was confirmedthrough a methanol desorption step of dyes adsorbed on the biomass,which was minimal.

The present experiment (FIG. 6) shows that the speed of this seconddecoloration, i.e. the transformation of the red intermediate product ina colorless product, is function of the availability of nutrients.During this experiment, the evolution of the absorbance at 500 nm wasstudied with cultures of the strain Pycnoporus sanguineus MUCL 38531containing different concentrations of malt. As shown on the graph inFIG. 6, the second decoloration of said red intermediate, was distinctlymore efficient when the culture medium contained the highestconcentration in malt.

Example 10

This example illustrates the color reduction that can be obtainedaccording to the process of the invention on real effluents. Thetreatment of industrial effluents using a process according to apreferred embodiment of the invention was studied. The effluents weresubmitted to ozone pretreatment, followed by a treatment with white-rotfungi.

Because of the complexity of the effluents to analyze, the inventorsproposed to characterize them, using four criteria a) their color(example 10, FIG. 7 and example 11, FIG. 13), b) their biodegradability(example 12, table 4), c) their toxicity on Caco2s human cells (example13, FIG. 8), which were used as model of the human intestinal epitheliumand d) the mutagenic character (example 14, FIGS. 9 to 12) of saideffluents before and after treatment.

Effluent samples from the dye industry Yorkshire Europe (previouslyCrompton and Knowles), were collected daily during 15 days. Thesesamples were mixed and constituted the crude effluent sample. Besides,some samples of ozone pretreated effluents were collected as describedabove, at same time. Each sample were diluted 5 times with a malt medium2% (w/v). The crude effluents or pretreated effluents samples wereinoculated with fragments of Pycnoporus sanguineus MUCL 41582. Theculture was incubated for 2 to 3 weeks at 25° C., at 125 rpm.

The effect of said treatments on the color of said effluents wasstudied. As shown FIG. 7, the treatment with ozone only decreased thecoloration of 30%, whereas the applied fungal treatment decreases thecolor of about 80%. The application of the process with ozonepretreatment and white-rot fungi treatment permits a reduction of thecoloration of about 90%.

Example 11

This example illustrates a pretreatment of liquid waste using wood chipsas an adsorption step. A sample of an industrial dye effluent wascollected and presents an initial color of 105000 color units (APHA).Wood chips were added and the pretreatment was applied during 24 hours.Color was measured (see “Ef+wood” in FIG. 13) and showed a decrease upto 72 000 APHA, which is less colored than the result obtained withozonolysis.

This pretreated effluent was then incubated in the presence ofPycnoporus sanguineus MUCL 41582 and 2 percent (w/v) malt during 1 week(see “Ef+wood+fungus” in FIG. 13) and compared with the same assaywithout pretreatment (see “Ef+fungus” in FIG. 13). Results showed thatthe pretreatment alone provoked a decoloration of 31%, and the fungaltreatment alone provoked a decoloration of only 5% in one week, whilethe combination enhanced the decolorization up to 74%. Moreover, asmentioned in example 10, two to three weeks were necessary to the fungusalone to decolorize the effluent in the same range as what is obtainedhere in one week with the combination proposed. Therefore, this systemallows to reduce by up to a third the contact time needed. Finally, asmentioned above the effects obtained by adsorption present the advantageof being considerably less expensive, since wood shavings areinexpensive.

Example 12

The problem with the effluents from dye or textile industries is thatthey are not biodegradable. Therefore, when they are dumped into abiological wastewater treatment plant, they are not degraded but justdiluted by other waters.

In this example, the biodegradability of effluents was measured beforeand after treatment of said effluents. The biodegradability of the crudeeffluent, the ozonised effluent, the crude effluent treated withwhite-rot fungi (crude Eff+WRF) and the ozonised effluent treated withwhite-rot fungi (O3+WRF Eff), was measured as the ratio BOD5/COD.

BOD is the abbreviation for the Biological or Biochemical Oxygen Demand.It is defined as the amount of oxygen (mg/l or mg/kg) used by thenon-photosynthetic microorganisms at 20° C. to metabolize biologicallydegradable organic compounds. Conventionally, we use the BOD₅, whichcorresponds to the amount of oxygen consumed after 5 days of incubation.The measurement of the parameters for the BOD₅ was done using amanometric method which relies on a difference of pressure and of thedecrease in pressure is measured by the Oxitop® system.

The measurement of the parameters for the COD was done through anormalized method (NFT 90.101 or DIN 38 409-H41-1). The method used wasa photometric determination of chromium (III) concentration after 2hours of oxidation with potassium dichromate/sulfuric acid/silversulfate at 148° C. A 620 nm filter was used.

The results are shown in following table 4. Ozonolysis does not allow toenhance biodegradability, nor does the fungal treatment used alone assuch (+7%). On the other hand, the combined process, i.e. ozonolysis andfungal treatment, doubles the biodegradability and is therefore moreefficient than the sum of any individual processes. TABLE 4 EffluentsBiodegradability Crude effluent 21% Ozonised effluent 20% crude Eff +WRF 28% O₃ + WRF Eff 41%

Example 13

The detoxifying effect of the process according to a preferredembodiment of the invention was studied by measuring the toxicity ofsaid effluents on Caco2 cells before and after treatments. Caco2 cellswere cultivated in Dulbecco's modified Eagle's medium (DMEM, Ref. 5796from Sigma) supplemented with 10% heat inactivated foetal bovine serum(Gibco) and 1% non-essential amino acids (Gibco). The cells wereincubated for 48 h at 37° C. Increasing concentration of the effluentswere tested on the Caco2 cells, a) crude effluent, b) ozone pretreatedeffluent, c) crude effluent treated with white-rot fungi and d) ozonepretreated effluent treated with white-rot fungi. After incubation for48 h at 37° C., the toxicity of the different effluents was measured bysubmitting the cells to a solution of tetrazolium salt (MTT,3(4,5-dimethylthiazolo-2-yl)-2,5-diphenyletrazolium bromide). Thetransformation of MTT to a red product, which is measured at 570 nm,gives indication on the production level of mitochondrial succinatedehydrogenase, enzyme which indicates the survival level of the cell.

The results obtained are presented FIG. 8, which shows a comparisonbetween the toxicity of the effluents (concentration 1%) on Caco2 cellsbefore and after ozone pretreatment and before and after treatment withwhite-rot fungi, wherein, crude Eff is crude Effluent, 03 Eff isozonised effluent, crude Eff+WRF is crude effluent treated withwhite-rot fungi and 03+WRF Eff is ozonised effluent treated withwhite-rot fungi.

It can be seen from this figure, that the ozonisation treatment onlyreduces the toxicity of 10%, whereas when the crude effluent is treatedwith white-rot fungi a 30% decrease of the toxicity is observed. Thebest results were obtained with ozone pretreated effluent which havebeen further treated with white-rot fungi, as a 70% decrease in toxicityis measured. This proves that the combination of ozone pretreatment withwhite-rot fungi treatment is more efficient than the sum of anyindividual processes.

Example 14

The mutagenicity of said effluents was studied before and aftertreatments. The tests of genotoxicity measure the capacity of a compoundto damage the DNA, this process being associated to the carcinogenesisof a compound. A method named VITOTOX™ has been developed, that uses theSOS response system of bacteria. The recombinant bacterial strains usedin this test contains different reporter genes, and are constructed withSalmonella typhimurium strains. The reporter system contains luciferasegene. In case of damage of the DNA of the strain rec2, the SOS responsesystem will be activated, as well as the luciferase gene. The luciferaseactivity can be measured by the light emission and is dependent of thegenotoxicity of the compound tested. The response measured is comparedto the one obtained in presence of another bacterial strain pr1. Ameasure wherein the response rec/pr1 is higher than 1,5 indicates themutagenicity of the sample. Moreover, when an organism meets anon-natural substance called xenobiotic, the liver transforms it tofacilitate the excretion by the kidneys. Therefore, some non-mutageniccompounds can be transformed into genotoxic products by metabolicactivation. For this reason, the test is coupled with an analysis of thesample to be tested after incubation with an exogenous metabolicextract. In our case, the mix S9/25 is a solution of hepatic hareextract which contains enzymes required for the detoxification of thexenobiotics ingested by the animal.

FIG. 9 shows the induction of Salmonella typhimudium SOS system(rec/pr1) according to the percentage of concentration in crude effluent(Crude Eff). FIG. 10 shows the induction of Salmonella typhimurium SOSsystem (rec/pr1) according to the percentage of concentration inozonised effluent (03 Eff). FIG. 11 shows the induction of Salmonellatyphimurium SOS system (rec/pr1) according to the percentage ofconcentration in Crude effluent treated with white-rot fungi (CrudeEff+WRF). FIG. 12 shows the induction of Salmonella typhimurium SOSsystem (rec/pr1) according to the percentage of concentration inozonised effluent treated with white-rot fungi (O3+WRF Eff).

By comparing FIGS. 9, 10, 11 and 12, it can be seen that the crudeeffluent is highly mutagenic, with or without metabolic activation (S9)for all the concentrations tested. The same can be observed for theozonised effluent without metabolic activation. With metabolicactivation the ozonised effluent is mutagenic from a concentration of2.5%. The crude and ozonised effluents which have been further treatedby white-rot fungi do not show any mutagenicity at the concentrationstested with or without metabolic activation. Such a result shows thedetoxifying effect of white-rot fungi on effluents from dye industries.The results of decoloration, biodegradability, toxicity and mutagenicityare summarized table 5. TABLE 5 Toxicity on Effluents ColorBiodegradability Caco2 Mutagenicity Crude effluent 100% 21% 100% yesOzonised effluent  71% 20%  90% yes crude Eff + WRF  24% 28%  69% noO3 + WRF Eff  9% 41%  29% no

From these results it can be seen that the process of the inventionpermits a 90% decrease of the color of industrial effluent, further morethe biodegradability of said effluent after treatment is doubled, 70% ofthe toxicity has been removed and the mutagenicity of said effluent hasbeen eliminated.

Example 15

The present example illustrates biochemical and performancecharacteristics of laccases excreted by highly efficientdye-transforming white-rot fungi. Selected white-rot fungi form thegroup consisting of Coriolopsis polyzona (MUCL 38443), Perenniporiaochroleuca (MUCL 41114), Pycnoporus sanguineus (MUCL 38531), Pycnoporussanguineus (MUCL 41582), Perenniporia tephropora (MUCL 41562), Trametesversicolor (MUCL 38412) and Clitocybula dusenii b11 (DSM 11238), wereused to produce laccases using draw-and-fill “Kefir principle” culture.Isoelectric points (pls), isozyme pattern of and effect of pH as well astemperature on the laccases were determined.

Table 6 summarizes the biochemical and performance characteristics oflaccases excreted by highly efficient dye-transforming white-rotbasidiomycetous fungi. TABLE 6 temperature pH stability [%] stability[%] (residual optimal (residual MW activity at temperature activity atoptimal (dimer) pl Fungal strain 70° C.) [° C.] pH 4/6) pH [kDa] (pH)Coriolopsis polyzona 9 40 19.4/396^(*)) 2 68 (147) 5.0, 5.2 Perenniporiaochroleuca 29 50   5/1379^(*)) 2 63 (144) 4.5, 5.0 Pycnoporus sanguineus58 50  2.7/45.8 2.5 70 4.4, 5.0, 5.3 MUCL 38531 Pycnoporus sanguineus 7050  0.5/617^(*)) 2.5 65 (136) 4.3 MUCL 41582 Perenniporia tephropora 7450 10.1/63.8 2 69 (148) 5.0, 6.2 Trametes versicolor 35 40  1.9/48.6 267 4.2, 4.5 Clitocybula dusenii 1 40  1.2/36.7 2.5 66 4.5, 4.7^(*))Activating effects were observed after incubation at pH 6 for 24 h.

Example 16

The decoloration of effluent by purified laccase enzymes from white-rotfungi with and without ozone pretreatment was studied. This experimentshows the role of ligninolytic enzymes of type laccases isolated fromwhite-rot fungi, in the decoloration of industrial effluent. Thisexperiment was conducted with industrial effluents contaminated withdyes. Samples from these effluents have been pretreated with ozone; theother samples constitute the crude effluents as collected on the site.The laccase used was isolated from a culture of Pyonoporus sanguineusMUCL 41582. They were concentrated by ultrafiltration and purified byanion-exchange chromatograpy, their molecular weight is 65 kDa and theirgene contain following sequences (primers used for the Polymerase ChainReaction: lac 1  5′ ACT GTG ACG GTC TTC GC 3′, lac 3: 3′ GTA GGT AAG GTAGAA GTG CCG 5′, lac 4: 5′ CAC TGG CAT GGC TTC TTC CA 3′, lac 5: 5′ AAGTCG ATG TGG CAG TGG AGG 3′)

a) with the primer lac 5 for the sequencing reaction:CCTGAAACCGGATGGTGACGTTGTCGCCGGGCGTGCCGGTGCTCACGACGTCGCGGAAGACCGGGTTGTCGTAGTTGTACTCGCTGCTTCCGGCGCTTCGGACGACAGCGAAGGTGTGCTGCAGTGATAGTGAGGTATAAATTTCTCATCAGATGCGTGATGAGCGGACGTACACCGTGCAGGTGGAAAGGGTGAGGGCTGCCAGGAGCATTGGCAGTGGCGGGGAAGGAGATCTCAATAGAGGCGTTGCTCGGCAGAACGTAGACGCTGCCTTCCGGGGACCAGGTCCTGTGCGGCCTGAGCGCCGCTGAGAATCTGGAGCAGGACCGGCACGGAGGGCGGGACGAAGGAGTGGTCATTGATGAAGAAGTTCGTGCCGTTCTATATGGACATCAGACAUGTCCCGAATTGTAGGACGCCGGTGCGAATGCTCACGAAGTTGAAAACCATGTTCAGCGGCTTGTCGACACCTCCGGGCTCGGGGCGGCCAGGCTATGATA TGATATATGGGTTTCACTC

b) with the primer lac 4 for the sequencing reaction:AGTCAACCAGTGCCCCATCGCTTCGGGCCATTCGTTCTTGTACGACTTCCAGGTCCCCGACCAAGCAGGTAATGAATTCGACACTCCCCTCCACTCGGTGATACTGACCCTTTTNAACTAGGAACNTTCTGGTATCACAGTCATCTATCCACCCAGTATTGTGATGGATTGAGAGGTCCCTTCGTCGTCTACGACCCGAATGATCCCCAGGCCAGCTTGTATGACAUGACAACGGTGAGCAGATTGGAGCCANGTCACATACTCTCTTCTTTCATACTGAAGCCACTCCCAGACGACACTGTGATTACTTTGGCCGACTGGTACCATCTTGCCGCTAAAGTTGGCCAGCGCTTCCCGTACGCCTTCTCCTATGTGTCTCGATGTTTCAAGTGGACTCATTGATGTGATGACAGAGTTGGCGCGGATGCGACTCTGATTAACGGGCTTGGTCGGACCCCCGGNACGACCTCTGCTGACCTGGCGATTATCAAGGTCACACAGGGCAAGCGGTTCGTGCCCATTTATCAACCTCTAATCGCTTGNCTCTGAC AATTCTGCTCTTT

c) with the primer lac inter 3 for the sequencing reaction: 5′ GGA ACTGGT TGC TCT GGC AGT ACG 3′

CGGGGACCAGGTCCTGTGCGGCCTGAGCGCCGCTGAGAATCTGGAGCAGGACCGGCACGGAGGGCGGGACGAAGGAGTGGTCATTGATGAAGAANTTCGTGCCGTTCTATATGGACATCAGACATTGTCCCGAATTGTAGGACGCCGGTGCGAATGCTCACGAAGTTGAAAACCATGTTCAGCGGCTTGTCGACACCTCCGGGCTCGGGGCGGCCAGGCTATGATATGATATATGGGTTTCA

Laccase from strain Pycnoporus sanguineus MUCL 41625 was also sequencedand gave following results:

a) with primer lac 5 for the sequencing reaction:ATNGTTGGTCTGAAACCGGATGGTGACGTTGTCGCCGGGGGTACCGGTGCTCACGACGTCACGGAAGACCGGGTTGTCGTAGTTGNACTCGCNGCTTCCGGCGCTTCGGACGACAGCGAAGGTGTGCTGCAGTGATTGTTAGAAATCAGCACGTACACCGTGCAGGTGGAAGGGGTGAGGGCTGCCAGGAGCATTGGCAGTGGCGGGGAAGGAGATCTCAATAGAGGCGTTGCTCGGGAGGACGTACACGCTGCCTTCCGGGACCAGGTCCTGTGCAGCCTGAGCGCCGCTGAGAATCTGGAGCAGGACCGGCACGGAGGGTGGGACGAAAGAGTGGTTGTTGATGAAGAAGTTCGTGCCGTTCTATATAGACATTAGACATTGTTCTAAACCATAGGACGCCGGCGGCGTATATACTCACGAAGTTGAAGACCATGTTGAGCGGCTTGGTCGACACCTCCGGGCTCGGGGCGTCCAGGCTATAATATGAGAGATTAGTTTTCATCTACTCGAATATTTANATCTGTACGAGAC

b) with primer lac 4 for the sequencing reaction:AGTCAACCAGTGCCCCATCGCTTCGGGCCATTCGTTCTTGTACGACTTCCAGGTTCCCGACCAAGCAGGTAACGAAATTTTGACACCCCCCTCCACTCGGTGATACTGATCCTTCTTTGATTAGGAACTTTCTGGTATCACAGTCATCTTTCCACCCAGTATTGTGATGGATTGAGAGGCCCCTTCGTCGTCTACGACCCGAATGATCCCCAGGCCAGCTTGTATGACATTGACAACGGTGAGTAATTTTGAGCCAAATGACACACTCTCTTGTCTTATACTGAAATCCCTACCAGACGACACTGTGATTACTTTGGCCGACTGGTACCATCTTGCCGCCAAAGTTGGACAGCGCTTCCCGTACGCCTTGTCTTATGTGTCTCGATGTTTCAAGTGGACTCATTGTCGTGATGACAGAGTTGGCGCGGATGCGACTCTGATTAACGGGCTGCTGATCTGGCGGTTATCAAGGTCACACAGGGCAAGCGGTTCGTGTCCAATATCAACTTATACTCGNTTTGCT

Laccase from strain Pycnoporus coccineus MUCL 38525 was also sequencedand gave following results:

a) with primer lac 5 for the sequencing reaction:CTGGCCGGTGCNCACGACGTCGCGGANGANCGGGTTATCGTAGTTGTACTCGNGGCTTCCGGAGCTTCGGACGACAGCGAAGGTGNGCTGNGTGTGATTGTTAGCTATCAACTCATCATCAGATGCGCGAGGACCGGACNTACACCGTGCAGGTGGAAGGGGTGAGGNGTGCCAGGAGCATTGGCAGTTGCGGGGAAGGAGATCTCAATAGACGAGTTGCTCGGGAGAACGTAGACGCTGCCGTCCGGAACGAGGTCCTGCGCGGCCTGAGCACCGCTGAGAATCTGGAGCAGAACCGGGACGGAGGGTGGGACAAAGGAGTGATTGTTGATGAAGAAGTTGGTGCCGTTCTGCAAGGACATCANANATTGTCCCAAAATGGCGCGAAGGCAATGCTCAC NAAGTTGAAAGACCATGTT

b) with primer lac 4 for the sequencing reaction:GTCAACCAGTGCCCCATCGCTTCGGGCCATTCGTTCTTGTACGACTTCCAGGTTCCCGATCAAGCAGGTAATGAAATTCGACCNGNTCTTTCATTCGGCG GGCCTGATCTC

The mixture enzymes/effluents has been incubated at 40° C. in a waterbath. The decoloration of said effluents has been measured after 24hours of incubation. The enzyme concentration was 2%. The decrease incoloration was measured after 1, 3 and 24 hours treatment. As shown inFIG. 14, the coloration of the sewage has been decreased visibly. It canbe also observed that the pretreatment with ozone permits to reach in 1hour a level of decoloration equivalent to the one obtained after 24hours of treatment in absence of said pretreatment.

Example 17

The following example illustrates the effect of immobilisation of activeagents in the treatment of dye-contaminated effluents. This exampledescribes the decoloration of an anthraquinonic dye by purified laccaseunder different conditions, i.e. used as a solution, or immobilised onwood chips.

One technique consisted of treating the dye solution with a solution oflaccases. One milliliter of NY3 dye at a concentration of 0.7 g/literwas treated with 100 microliter of laccase concentrated from Pycnoporussanguineus MUCL 41582 during 24 hours at 40° C.

A second technique consisted of immersing wood chips in a laccasesolution. One milligram of wood shaving was immersed during two secondsin the same concentrated laccase. The piece of wood containing adsorbedlaccase was then immersed into one milliliter of NY3 dye at aconcentration of 0.7 g/liter and incubated during 24 hours at 40° C. Thereaction was followed spectrophotometrically and results are shown inFIG. 15.

It can be observed that the initial dye has completely been decolorizedin the two techniques since an absorbance peak at the initial 595 nmcould not be detected anymore. This result was due to a transformation,not to adsorption as indicated by the spectral change (new peak at 500nm, as observed previously in example 9, FIG. 5). Nevertheless, thetransformation was more complete when the laccase was adsorbed on woodas an absorbance of 0.2 instead of 0.45 was observed when the laccasewas added as a solution.

A simple and inexpensive way to extract, immobilize and concentrateactive agent comprises the following process. A white-rot fungus iscultured in a liquid medium until enzyme production has reached asignificant amount. Then a support, preferably a wooden support such aswood chips is added the supernatant. The enzymes released in theextracellular medium can then easily be adsorbed on this wood support.Such method enables to work with the enzymes produced by the fungiinstead of working with a complete fungal culture.

Such method also avoids the need to concentrate and purify the enzymesout of the cultures by means of highly expensive and complicatedtechniques. The above-described method also enables to limit the costs,and provides a method, which can be used in industrial processes for theenvironmental treatments.

1. A process for the treatment of liquid waste, comprising the steps of:(a) submitting said liquid waste to a pretreatment; and (b) submittingsaid pretreated liquid waste to the action of white-rot fungi or activeagents thereof.
 2. The process according to claim 1, wherein saidwhite-rot fungi active agents comprise hydrolytic enzymes, cellulolyticenzymes, or ligninolytic enzymes.
 3. The process according to claim 2,wherein said white-rot fungi active agents consist essentially oflaccase enzymes.
 4. The process according to claim 1, wherein saidpretreatment is ozonizsation.
 5. The process according to claim 1,wherein said pretreatment comprises adsorption of said waste on abiodegradable support.
 6. The process according to claim 1, wherein saidliquid waste, is dye-containing liquid waste, comprising azo dyes andanthraquinones dyes
 7. The process according to claim 1, wherein saidliquid waste comprises humic acids.
 8. The process according to claim 1,wherein said white-rot fungi are selected from the group consisting ofthe genus Acanthophysium, the genus Aleurobotrys, the genusAleurodiscus, the genus Amphinema, the genus Amylostereum, the genusArmillaria, the genus Aspergillus, the genus Asterostroma, the genusAuricularia, the genus Botryobasidium, the genus Botryohypochnus, thegenus Calocera, the genus Chaetomium, the genus Cladorrhinum, the genusClitocybula, the genus Columnocystis, the genus Coriolopsis, the genusCystostereum, the genus Daedalea, the genus Daedaleopsis, the genusDichomitus, the genus Dichostereum, the genus Echinodontium, the genusFibulomyces, the genus Fomitopsis, the genus Fusarium, the genusGanoderma, the genus Grifola, the genus Hapalopilus, the genus Humicola,the genus Hymenochaete, the genus Hyphoderma, the genus Hyphodontia, thegenus Hypochnicium, the genus Inonotus, the genus Irpex, the genusLaurilia, the genus Laxitextum, the genus Lentinus, the genus Lenzites,the genus Lentinula, the genus Leucogyrophana, the genus Lycoperdon, thegenus Marasmius, the genus Merulius, the genus Mycoacia, the genusMyrothecium, the genus Paecilomyces the genus Panellus, the genusPenicillium, the genus Peniophora, the genus Perenniporia, the genusPestalolia, the genus Phanerochaete, the genus Phellinus, the genusPhlebia, the genus Pholiota, the genus Pleurotus, the genus Polyporus,the genus Poria, the genus Punctularia, the genus Pycnoporus, the genusResinicium, the genus Schizophyllum, the genus Scytinostroma, the genusSteccherinum, the genus Trametes, the genus Trichoderma, the genusTyromyces and the genus Vararia.
 9. The process according to claim 1,wherein said white-rot fungi are selected from the group consisting ofthe genus Acanthophysium, the genus Coriolopsis, the genus Clitocybulathe genus Cystostereum, the genus Ganoderma, the genus Paecilomyces, thegenus Perenniporia, the genus Phellinus, the genus Phlebia, the genusPycnoporus and, the genus Trametes.
 10. The process according to any ofclaim 1, wherein said white-rot fungi are selected from the groupconsisting of Acantophysium bisporum MUCL 32213, Coriolopsis polyzonaMUCL 38443, Cystostereum murraii MUCL 33747, Ganoderma subamboinenseMUCL 38859, Lentinus cladopus MUCL 28678, Lentinula edodes MUCL 29756,Lenzites betulina MUCL 38559, Merulius tremelosus MUCL 38065,Paecilomyces variotii MUCL 21705, Perenniporia medulla-panis MUCL 40050,Perenniporia ochroleuca MUCL 41114, Perenniporia tephropora MUCL 41562,Phanerochaete chrysosporium MUCL 19343, Phanerochaete ericina MUCL33845, Phellinus rimosus MUCL 38446, Phlebia subserialis MUCL 33724,Polyporus brumalis MUCL 29280, Polyporus ciliatus MUCL 40141, Pycnoporuscinnabarinus MUCL 38520, Pycnoporus coccineus MUCL 38525, Pycnoporussanguineus MUCL 41625, Trametes versicolor MUCL 38412 and MUCL 28407.11. The process according to claim 1, wherein said white-rot fungi areselected from the group consisting of Clitocybula dusenii DSM 11238,Trichoderma harzanium MUCL 29707 and Trichoderma longibrachiatum MUCL39887.
 12. The process according to claim 1, wherein said white-rotfungi are grown in a media containing malt extract in a concentrationranging from 0.5 to 8 percent by weight to volume.
 13. The processaccording to claim 1, wherein said white-rot fungi are added to saidliquid waste in an encapsulated form, in a matrix consisting ofpolymers.
 14. The process according to claim 13, wherein said polymersare selected from the group consisting of alginate salts, carrageenansalts, iota-carrageenan salts, maltodextrin, whey protein concentrate(WPC), skimmed milk powder (SMP), dried yeast autolysate (YA), driedyeast extract (YE), corn starch (CS), modified starch (MS), andpolyvinylalcohol.
 15. The process according to claim 1, wherein saidwhite-rot fungi are employed in an immobilized form.
 16. The processaccording to claim 15, wherein said white-rot fungi are immobilized on asupport selected from the group consisting of stainless steel support,polymer support and wood support.
 17. The process according to claim 1,wherein the white-rot fungi active agents are employed as rawpreparation, as purified enzymes, or in an immobilized form.
 18. Theprocess according to claim 17, wherein the white-rot fungi active agentsare immobilized on a wood support.
 19. The process according to claim 1,further comprising adding an inductor to white-rot fungi culture,preferably after said fungi has reached a significant bio-mass.
 20. Theprocess according to claim 19, wherein said inductor has an azoanthraquinonic or a stilbenic dye structure.
 21. The process accordingto claim 19, wherein said inductor has a phenolic, aromatic or metallicstructure.
 22. The process according to claim 1, further comprisingincubating said white-rot fungi in said pretreated liquid waste.
 23. Theprocess according claim 22, wherein a suitable amount of nutrients areadded during the incubation of said white-rot fungi in said pretreatedliquid waste.
 24. The process according to claim 23, wherein thenutrients are added in an amount ranging from 0.5 to 4 percent in weightper volume.
 25. The process according claim 22, wherein a pH during theincubation of said white-rot fungi in said pretreated liquid waste isranging from 4 to 9 and a temperature of incubation is ranging from 20to 45° C.
 26. The process according to claim 22, wherein a pH during theincubation of said active agents in said pretreated liquid waste isranging from 2 to 7 and a temperature of incubation is ranging from 20to 70° C.
 27. Ihe process according to claim 22, wherein a pH during theincubation of said white-rot fungi or active agents thereof in saidpretreated liquid waste is ranging from 1 to 9 and a temperature ofincubation is ranging from 18 to 70° C.
 28. The process according toclaim 22, wherein said white-rot fungi or active agents thereof, areincubated with said pretreated liquid waste for 2 hours to 14 days. 29.The process according to claim 22, wherein said white-rot fungi oractive agents thereof, are incubated with said pretreated liquid wastefor 20 minutes to 14 days.
 30. The process according to claim 1, whereinsaid fungi or active agents thereof obtainable after step b) areseparated.
 31. The process, according to claim 30, wherein saidseparated fungi or active agents thereof are reused in said process. 32.A method of using the fungi or active agents thereof obtainable afterstep b) of the process according to claim 1 into a green wastecomposting process.
 33. A method for immobilizing fungal active agentson a support comprising the steps of: culturing a fungus in a liquidmedium, immersing a support with the supernatant of said fungal culture,and immobilizing the active agents of said fungi on said support, saidagents being released in the fungal culture supernatant.
 34. The methodaccording to claim 33 wherein said support comprises a wood support. 35.The method according to claim 34, wherein said wood support is immersedwith said fungal culture supernatant in a quantity comprised between 0.1and 100 g per liter of fungal culture supernatant.
 36. The methodaccording to claim 34, wherein said wood support is immersed in saidfungal culture supernatant from 1 second to 24 hours.
 37. The processaccording to claim 22, wherein oxygen is added during the incubation ofsaid white-rot fungi in said pretreated liquid waste.